Development and Applications of Bioorthogonal Chemistry ABSTRACT A major hurdle in biophysical studies of class B GPCR conformational transitions, particularly the movements of the two large domains during ligand-induced activation, is that there are very few techniques available that allow site-specific introduction o biophysical probes into these two domains without altering the receptor function. To overcome this limitation, our long-term goal is to develop bioorthogonal chemistry tools that enable biophysical studies of the multi-domain signaling proteins such as class B GPCRs in living cells. In our previous studies, we have optimized a bioorthogonal, photoinduced tetrazole-alkene cycloaddition reaction ('photoclick chemistry'), and developed the palladium-mediated cross-coupling reactions for selective protein labeling in living systems as well as a phage-assisted interrogation of reactivity strategy for evolving the sequence-specific bioorthogonal reactions. Built upon these results, in this application we plan to integrate the bioorthogonal chemistry tools with the genetic encoding of unique chemical functionalities to generate in situ the chemically modified GLP-1R/GCGR?two members of the class B GPCRs that are implicated in diabetes and obesity, and study their conformational transitions and photo-regulation in living cells. The specific aims are as follows: 1) Apply photoclick chemistry to generate in situ the environment-sensitive fluorescent probes on GCGR/GLP-1R and probe the ligand-induced conformational changes in living cells. A spiro[2,3]hex-1-ene or fumarate-derived lysine will be site-specifically incorporated at the extracellular loop 3 region of GCGR/GLP- 1R to direct the photoclick chemistry, and the resulting fluorescent labeled GCGR/GLP-1R will be used in the studies of the conformational transitions induced by the specific ligands; 2) Develop binary bioorthogonal chemistry for dual-labeling of GCGR/GLP-1R to probe the ligand-induced conformational changes by FRET in living cells. The photoclick chemistry will be used in tandem with the sequence-specific palladium-mediated cross-coupling or the cysteine-nitrile condensation reaction to enable the simultaneous introduction of two fluorophores at the juxtamembrane domain and the N-terminal extracellular domain, respectively. The dynamic movements of these two interconnected domains upon perfusion of the specific peptide ligand in living cells will be monitored by FRET using confocal microscope; 3) Develop the azobenzene-based optochemical genetic tools for optical regulation of GLP-1R activation in living cells. A biocompatible inverse azo-coupling reaction based on the condensation of phenylhydrazines with fluoroquinols will be developed, which together with the genetic encoding of fluoroquinolalanine, will allow us to introduce the azobenzenes site-specifically into the two regulatory regions of GLP-1R. The effect of reversible photoswitching on GLP-1R activity in the absence or presence of GLP-1 will be assessed using b-arrestin-mCherry as a reporter for receptor activation. These studies will provide the key insights into GLP-1R/GCGR activation mechanisms, which are crucial for the development of GLP-1R/GCGR dual agonists as therapeutic agents for the treatment of diabetes and obesity.